Huaxia Xin

Purification of flavonoids from licorice using an off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method

An orthogonal (71.9%) off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self-made Click TE-Cys (60 μm) solid-phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE-Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co-eluted in the first dimension were selected for further purification using reversed-phase liquid chromatography. Multiple compounds could be isolated from one normal-phase fraction and some compounds with bad resolution in one-dimensional liquid chromatography could be prepared in this two-dimensional system owing to the orthogonal separation. Moreover, this two-dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off-line two-dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice.

Hydrophilic interaction liquid chromatography for the separation, purification, and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz

A systematic strategy based on hydrophilic interaction liquid chromatography was developed for the separation, purification and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz. Methods with enough hydrophilicity and selectivity were utilized to resolve the problems encountered in the separation of oligosaccharides such as low retention, low resolution and poor solubility. The raffinose family oligosaccharides in L. lucidus Turcz. were isolated using solid-phase extraction followed by hydrophilic interaction liquid chromatography at semi-preparative scale to obtain standards of stachyose, verbascose and ajugose. Utilizing the obtained oligosaccharides as standards, a quantitative determination method was developed, validated and applied for the content determination of raffinose family oligosaccharides both in the aerial and root parts of L. lucidus Turcz. There were no oligosaccharides in the aerial parts, while in the root parts, the total content was 686.5 mg/g with the average distribution: raffinose 66.5 mg/g, stachyose 289.0 mg/g, verbascose 212.4 mg/g, and ajugose 118.6 mg/g. The result provided the potential of roots of L. lucidus Turcz. as new raffinose family oligosaccharides sources for functional food. Moreover, since the present systematic strategy is efficient, sensitive and robust, separation, purification and quantification of oligosaccharides by hydrophilic interaction liquid chromatography seems to be possible.

Development, validation and application of a hydrophilic interaction liquid chromatography-evaporative light scattering detection based method for process control of hydrolysis of xylans obtained from different agricultural wastes

Purified standards of xylooligosaccharides (XOSs) (DP2-6) were first prepared from a mixture of XOSs using solid phase extraction (SPE), followed by semi-preparative liquid chromatography both under hydrophilic interaction liquid chromatography (HILIC) modes. Then, an accurate quantitative analysis method based on hydrophilic interaction liquid chromatography-evaporative light scattering detection (HILIC-ELSD) was developed and validated for simultaneous determination of xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6). This developed HILIC-ELSD method was applied to the comparison of different hydrolysis methods for xylans and assessment of XOSs contents from different agricultural wastes. The result indicated that enzymatic hydrolysis was preferable with fewer by-products and high XOSs yield. The XOSs yield (48.40%) from sugarcane bagasse xylan was the highest, showing conversions of 11.21g X2, 12.75g X3, 4.54g X4, 13.31g X5, and 6.78g X6 from 100g xylan.

Purification of Lignans from Fructus Arctii using off-line two-dimensional supercritical fluid chromatography/reversed-phase liquid chromatography

As a common traditional Chinese medicine, Fructus Arctii has important clinical medical values. Its main components are lignans, which are difficult to separate and analyze because of the complex composition, similar chemical structures, and close properties. In this study, an off-line two-dimensional supercritical fluid chromatography/reversed-phase liquid chromatography method, as well as an effective sample pretreatment method based on hydrophilic interaction chromatography material, was developed to enrich the minor lignan fractions and obtain high-purity compounds. In total, 12 high-purity compounds were isolated from Fructus Arctii. Their structures were identified by using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, which showed that all were lignans and that most of them were isomers. The results demonstrated the effective off-line two-dimensional supercritical fluid chromatography/reversed-phase liquid chromatography method for the purification of lignans from Fructus Arctii. The separation protocol established here will be beneficial for the separation of complex samples from other kinds of natural products.

Rapid purification of diastereoisomers from Piper kadsura using supercritical fluid chromatography with chiral stationary phases

Supercritical fluid chromatography (SFC) with chiral stationary phases (CSPs) is an advanced solution for the separation of achiral compounds in Piper kadsura. Analogues and stereoisomers are abundant in natural products, but there are obstacles in separation using conventional method. In this paper, four lignan diastereoisomers, (-)-Galbelgin, (-)-Ganschisandrin, Galgravin and (-)-Veraguensin, from Piper kadsura were separated and purified by chiral SFC. Purification strategy was designed, considering of the compound enrichment, sample purity and purification throughput. Two-step achiral purification method on chiral preparative columns with stacked automated injections was developed. Unconventional mobile phase modifier dichloromethane (DCM) was applied to improve the sample solubility. Four diastereoisomers was prepared at the respective weight of 103.1mg, 10.0mg, 152.3mg and 178.6mg from 710mg extract with the purity of greater than 98%.

Selective separation of xanthones and saponins from the rhizomes of Anemarrhena asphodeloides by modulating the density of surface charges in C18-bonded stationary phases

A rapid and robust separation method based on the positively charged reversed-phase (PGRP) stationary phase was developed for selective separation of saponins and xanthones from the rhizomes of Anemarrhena asphodeloides (A. asphodeloides). In this work, the chromatographic performances of three PGRP stationary phases with different surface positive charge densities were systematically evaluated by studying hydrophobicity, effects of pH and buffer concentration of the mobile phase, ion-exchange capacity, etc. The PGRP stationary phase exhibited reversed-phase/anion-exchange mixed-mode properties. Then the retention behaviors of xanthones were investigated. A good retention of xanthones was obtained at high pH and xanthones were easily eluted at low pH. The pH is like an on–off switch on the PGRP stationary phase that controls the retention of xanthones. Finally, this PGRP material was successfully applied to the selective separation of saponins and xanthones from A. asphodeloides. The result demonstrated that neutral and ionic compounds, such as xanthones and saponins from the rhizomes of A. asphodeloides, were separated selectively by modulating both the density of surface charges in the PGRP stationary phase and the pH of the mobile phase.

Separation of Piper kadsura Using Preparative Supercritical Fluid Chromatography Combined with Preparative Reversed-Phase Liquid Chromatography

We have developed a separation strategy that employed preparative supercritical fluid chromatography (prep-SFC) combined with preparative reversed-phase liquid chromatography (prep-RPLC) to purify compounds from Piper kadsura. The complexity of the sample was effectively reduced by two steps of prep-SFC. The key experimental parameters of prep-SFC were systematically optimized, including an “average-pressure rule” scaling-up method for adjustment of the backpressure of the system. A mixed modifier consisting of ethanol/n-hexane was applied to provide a stable elution. Prep-RPLC was subsequently adopted to purify the SFC fractions due to its good orthogonality with SFC. Furthermore, chiral SFC was used to separate the structural analogues. As a result, 14 compounds with high purity were obtained from one fraction, including three pairs of diastereoisomers. These data indicate that the separation strategy using prep-SFC combined with prep-RPLC is an effective tool to separate complex samples such as natural products.

Preparative separation of the polar part from the rhizomes of Anemarrhena asphodeloides using a hydrophilic C18 stationary phase

The goal of this study was to develop a method that utilized a hydrophilic C18 stationary phase in the preparative high performance liquid chromatography to isolate the polar part from the rhizomes of Anemarrhena asphodeloides. The results showed that an initial mobile phase of pure water for the separation could greatly increase the retention and solubility of the polar compounds at the preparative scale. Introducing polar groups on the surface of the hydrophilic C18 column together with the use of optimized mobile phase compositions improved the column separation selectivity for polar compounds. Eleven previously undescribed compounds in Anemarrhena asphodeloides were obtained, indicating that the method developed in this study would facilitate the purification and separation of the polar part of traditional Chinese medicines.

Separation and purification of the components in Trachelospermum jasminoides by two dimensional hydrophilic interaction liquid chromatography- reversed-phase liquid chromatography

A preparative two dimensional hydrophilic interaction liquid chromatography/reversed-phase liquid chromatography (Pre-2D-HILIC/RPLC) method was established to separate and purify the components in Trachelospermum jasminoides. The pigments and strongly polar components were removed from the crude extract after the active carbon decolorization and solid phase extraction processes. A Click XIon column (250 mm×20 mm, 10 μm) was selected as stationary phase and water-acetonitrile as mobile phases in the first dimensional HILIC. Finally, 15 fractions were collected under UV-triggered mode. In the second dimensional RPLC, a C18 column (250 mm×20 mm, 5 μm) was selected and water-acetonitrile was used as mobile phases. As a result, 14 compounds with high purity were obtained, which were further identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Finally, 11 lignan compounds and three flavonoid compounds were obtained. The method has a good orthogonality, and can improve the resolution and the peak capacity. It is significant for the separation of complex components from Trachelospermum jasminoides.

Fingerprint profiling of Astragalus polysaccharides based on partial acid hydrolysis-hydrophilic interaction chromatography analysis and comprehensive quality evaluation of Astragalus membranaceus combined with reversed-phase liquid chromatography fingerprint analysis

Astragalus polysaccharides (APS) are important components in Astragalus membranaceus ( A. membranaceus ). However, for the huge relative molecular mass, strong polarity, and difficulty in chromatographic separation, theres a lack of quality control finely reflecting the composition of APS up to now. To solve the problems, the key lies in degradation of polysaccharides and analysis of the hydrolyzates. A partial acid hydrolysis-hydrophilic interaction liquid chromatography (HILIC) fingerprint for APS and a comprehensive quality evaluation method for A. membranaceus combining with the traditional RPLC fingerprint were developed. Orthogonal experiments were designed to investigate the influencing factors of partial hydrolysis and the conditions were optimized based on HILIC analysis. The hydrolyzates were successfully separated using HILIC-evaporative light scattering detection (ELSD) method and the optimal conditions of hydrolysis were selected as 80℃, 1.5 mol/L trifluoroacetic acid and 4 h, which proved to be stable and repeatable after the validation. This method was applied for the fingerprint analysis of 20 batches of A. membranaceus . Cosine method was used for similarity calculation using the average of samples Nos. 1-10 as reference. Moreover, RPLC fingerprint analysis was processed on other components and the data were compared for comprehensive evaluation. The similarities of ten samples with the reference ranged from 0.258 to 0.949, which were consistent with the chromatograms reflecting the apparent disparity in APS, and could be as a supplement of the RPLC fingerprint. The new method, with the advantages of intuition, convenience, and quantifiable in similarity, could reflect the differences of the composition of polysaccharides after degradation and was successfully applied in profiling of APS, contributing to the quality control together with the RPLC fingerprint.